| Objective:Optogenetics is a novel neuromodulation technology that uses light-sensitive channel protein to specifically activate or inhibit neuronal activity.This study aimed to explore the improvement of optogenetics neuromodulation on working memory dysfunction of vascular dementia(VD)rats by activating parvalbumin(PV)interneurons in the medial prefrontal cortex.Furthermore,to reveal the possible mechanism that PV interneurons affect the excitatory and inhibitory balance of medial prefrontal cortex in VD rats.Methods:(1)Forty healthy male SD rats of SPF grade were divided into sham operation group with ten rats and operation group with thirty rats according to the random number table method.For the operation group,bilateral common carotid artery permanent ligation surgery performed to construct the model of VD rats according to Kazuo SUGIO method.In contrast,the sham operation group was only separated the bilateral common carotid arteries without ligation.Three weeks before modeling,the operation group injected with ChR2 optogenetics virus using PV interneurons as promoters in medial prefrontal cortex and then implanted with optical fibers.The 3D-MRA angiography test used to detect the success of bilateral common carotid artery permanent ligation,and the Y maze and novel object recognition test were used to test the behaviors.(2)Rats with successful modeling divided into three groups according to the random number stable method:the model group,the optogenetics intervention group,and the sham optogenetics intervention group,ten rats in each group.After two weeks of modeling,optogenetics intervention and optogenetics sham intervention carried out.The optogenetics intervention group used 473 nm wavelength light stimulation with a frequency of 40 Hz,a light intensity of 1 mW,a stimulation interval of 5 ms,lasting 15 min,and once a day,for a total of 3 weeks.The sham optogenetics intervention group used 550 nm wavelength light to stimulation,and other parameters were consistent with the optogenetics intervention group.The sham operation group and the model group grabbed under the same conditions without intervention.(3)Magnetic resonance spectroscopy imaging used to analyze the change characteristics of neurochemical metabolism,such as excitatory neurotransmitter glutamate(Glu),inhibitory neurotransmitter γ-aminobutyric acid(GABA)and N-acetyl aspartate(NAA).(4)Y-maze spontaneous alternation experiment used to test the working memory of rats in each group.(5)Laser confocal scanning microscopic imaging used to observe the expression of PV interneurons in the medial prefrontal cortex.Nissl staining used to observe the neuronal morphology and neuron loss in the medial prefrontal cortex;Immunohistochemistry used to analyze the expression of GAD67 and NMDAR in the medial prefrontal cortex.Results:(1)Vascular dementia model test:① Angiography imaging showed that the blood flow in the brain of the sham operation group was smooth,the blood vessels were visible,and there was no apparent abnormal shape.After modeling,the rats of the model group,the optogenetics intervention group,and the sham optogenetics intervention group showed apparent vascular interruption.② The results of Y maze alternation experiment and novel object recognition test showed that the preference index of novel objects decreased for 1 hour of the model group,the optogenetics intervention group,and sham optogenetics intervention group after two weeks of modeling(P<0.001),spontaneous alternation rate of Y maze also decreased(P<0.05 or P<0.01).Moreover,there was no statistical difference among the three groups(P>0.05),which indicates that vascular dementia rats had working memory dysfunction and novel object recognition disfunction at 14 days after modeling.The three groups of rats had the same baseline before intervention.(2)The expression of light-sensitive channel protein in PV interneurons of the medial prefrontal cortex:PV interneurons expressed green fluorescence,which indicates that the PV interneurons in the medial prefrontal cortex of the two groups successfully expressed the light-sensitive channel protein.(3)Effect of optogenetics regulation of PV interneurons in the medial prefrontal cortex in vascular dementia rats with working memory:Compared with the sham operation group,the Y maze alternation rate of the model group was significantly reduced(P<0.01),and compared with the model group and the sham optogenetics intervention group,the alternation rate of the optogenetics intervention group was significantly increased(P<0.01),and the mean difference showed that after the intervention,compared with the model group and the sham optogenetics intervention group,the alternation rate of the optogenetics intervention group was significantly higher than before(P<0.05 or P<0.01),suggesting that optogenetics can effectively improve the working memory impairment of vascular dementia rats by regulating the PV interneurons of medial prefrontal.(4)Effect of optogenetics regulated of PV interneurons in the medial prefrontal cortex on neurotransmitter metabolites in vascular dementia rats:In the model group,the content of NAA in the medial prefrontal cortex decreased(P<0.001),the content of GABA inhibitory neurotransmitter decreased(P<0.05),while the content of Glu the content of GABA inhibitory neurotransmitter increased(P<0.05),compared with the sham operation group.The content of NAA in the medial prefrontal cortex increased(P<0.05 or P<0.01),the content of GABA increased(P<0.05),and there was no statistically significant change in Glu content.However,there was a decreasing trend(P>0.05)between them.It indicated that optogenetics regulation could promote the balance of neurotransmitter release in the medial prefrontal cortex of vascular dementia rats.(5)Effect of optogenetics regulated PV interneurons in the medial prefrontal cortex on histopathology in vascular dementia rats:Nissl staining showed that the Nissl bodies in the medial prefrontal neurons of the sham operation group enriched.The nerve cells were densely and neatly arranged,with a large number,and in the model group,neurons scattered and lightly stained,and the number significantly reduced.The number of medial prefrontal nerve cells in the optogenetic intervention group increased.The optogenetic sham intervention group did not show changes in the number of medial prefrontal Nissl bodies.(6)Effect of optogenetics regulated PV interneurons in the medial prefrontal cortex on the expression of GAD67 and NMMDAR in vascular dementia rats:Compared with the sham operation group,the expression of GAD67 reduced(P<0.01 or P<0.001)and the expression of glutamate receptor NMDAR increased(P<0.05 or P<0.01)in the medial prefrontal cortex of the model group.Compared with the model group and sham optogenetics intervention group,the expression of GAD67 increased(P<0.05),and the expression of NMDAR decreased(P<0.05)in the medial prefrontal cortex of the optogenetics intervention group.It indicates that optogenetics could regulate the expression of GABA and Glu receptors in the medial prefrontal cortex of vascular dementia rats.Conclusion:Optogenetics regulated PV interneurons in the medial prefrontal cortex could improve working memory dysfunction in vascular dementia rats,which is related to the activation of PV interneurons to regulate the release of GABA/Glu neurotransmitters and their receptors expressions that promoted excitation and inhibition balance of medial prefrontal cortex. |
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